Surface for the immobilisation of nucleic acids

ABSTRACT

The invention relates to a surface for the immobilization of one or several first nucleic acids as recognition elements (“immobilization surface”), for the production of a recognition surface for the detection of one or several second nucleic acids in one or more samples which are brought into contact with the recognition surface, the first nucleic acids being applied to a layer of the graft copolymer poly(L-lysine)-g-poly(ethyleneglycol) (PLL-g-PEG) as surface for immobilization, characterized in that the grafting ratio g, in other words the ratio between the number of lysine units and the number of polyethylene glycol side chains (“PEG” side chains) has an average value between 7 and 13. The invention also relates to a method for the qualitative and/or quantitative detection of one or more second nucleic acids in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with an immobilization surface according to the invention, upon which one or several first nucleic acids are immobilized as recognition elements for specific binding/hybridization with said second nucleic acids and changes in optical or electronic signals resulting from the binding/hybridization of said second nucleic acid, or further, resulting from applied tracer substances applied for analyte detection, are measured.

The invention in hand relates to a surface for the immobilization of oneor several first nucleic acids as recognition elements (“immobilizationsurface”), for the production of a recognition surface for the detectionof one or several second nucleic acids in one or more samples which arebrought into contact with the recognition surface, the first nucleicacids being applied to a layer of the graft copolymerpoly(L-lysine)-g-poly(ethyleneglycol) (PLL-g-PEG) as surface forimmobilization, characterized in that the grafting ratio g, in otherwords the ratio between the number of lysine units and the number ofpolyethylene glycol side chains (“PEG” side chains) has an average valuebetween 7 and 13. In the following, the “immobilization surface” asdefined above, together with the first nucleic acids immobilizedthereon, is called a “recognition surface”.

The invention also relates to a method for the qualitative and/orquantitative detection of one or more second nucleic acids in one ormore samples, characterized in that said samples and optionally furtherreagents are brought into contact with an immobilization surfaceaccording to the invention, upon which one or several first nucleicacids are immobilized as recognition elements for specificbinding/hybridization with said second nucleic acids and changes inoptical or electronic signals resulting from the binding/hybridizationof said second nucleic acid, or further, resulting from applied tracersubstances applied for analyte detection, are measured.

In the context of the invention in hand, the term “nucleic acids” shallmean single- or double-stranded compounds from the group formed byoligonucleotides, polynucleotides, DNA or RNA strands and DNA or RNAanalogs, e.g. comprising modified bases or “backbones”. In thisdefinition of the term “nucleic acids” shall also be included hybrids ofDNA and RNA and their analogs.

For the detection of one or more analytes from a sample with a complexmixture of numerous substances there are widespread methods in which oneor more so-called recognition elements which are of biological,biochemical or synthetic character are immobilized on a solid carrierbefore they are then brought into contact in immobilized form with saidsample and the analytes contained therein bind to the recognitionelements specific for them. In this case, the solid carrier may be bothof macroscopic nature with a surface of square millimeters to squarecentimeters or also of microscopic nature, for example in the form ofso-called beads, i.e. approximately spherical particles with typicaldiameters in the micrometer range. The surface of such a solid carrierwith recognition elements immobilized thereon shall hereinafter becalled a “recognition surface”.

Compared with methods in which the analytes and their recognitionelements are brought together as reaction or binding partners inhomogeneous liquid solution, these methods which are based on a solidcarrier offer numerous advantages, for example an easier separation ordifferentiation of bound analyte molecules from the sample matrix. Theseadvantages are gained with a restriction of the diffusion-driven mixturebetween analyte molecules and recognition elements, because of thebinding of the recognition elements to the solid carrier.

For the preparation of recognition surfaces for the highly efficient andhighly selective binding of the one or more analytes to be detected in asample, the quality of these surfaces is of major importance. To achievethe lowest possible limits of detection, it is desirable to immobilizein a small space as many recognition elements as possible in such a waythat as many analyte molecules of one variety as possible may then bebound in the later detection process. At the same time it is desirableon immobilization to maintain as high a degree of reactivity andbiological or biochemical functionality of the recognition elements aspossible, i.e. to minimize any signs of denaturation resulting from theimmobilization. A further objective is as far as possible to prevent thenonspecific binding or adsorption of analyte molecules which in manycases have the effect of restricting the limits of detection attainable.

Especially for the analysis of nucleic acids, microarrays with apartially very high “feature density”, i.e. density of discretemeasurement areas comprising biological or biochemical recognitionelements immobilized therein on a common carrier, are known since about1990.

Within the terms of the present invention, laterally separatemeasurement areas, as an integral part of a recognition surface, shallbe defined by the surface area which encompasses the biological orbiochemical or synthetic recognition elements immobilized thereon forthe detection of an analyte from a liquid. These areas may be present inany geometric form, for example in the form of points, circles,rectangles, triangles, ellipses or lines. It is possible that up to1,000,000 measurement areas may be present in a two-dimensionalarrangement, wherein a single measurement area may take up an area of0.001 mm²-6 mm². The density of the measurement areas may typicallyamount to more than 10, preferably more than 100, especially preferablymore than 1000 measurement areas per square centimeter.

In the following, an array shall mean a two-dimensional arrangement ofmeasurement areas on a common carrier. Thereby, the carrier may have anessentially planar or also any other, for example spherical geometry.

In U.S. Pat. No. 5,445,934 (Affymax Technolgies), for example, arrays ofoligonucleotides arranged at a density of more than 1000 features persquare centimeter are described and claimed.

For an improvement of the adhesion and stability of the immobilizationof biological, biochemical or synthetic recognition elements it is oftenadvantageous to deposit initially a so-called adhesion-promoting layeron the carrier. The adhesion-promoting layer may comprise, for example,chemical compounds compound from the group of silanes, functionalizedsilanes, epoxides, functionalized, charged or polar polymers and“self-assembled passive or functionalized monolayers or multilayers”.Such adhesion-promoting layers and specific requirements on theproperties of an adhesion-promoting layer, which are dependent on thephysical and chemical type of the carrier and the related measurementarrangement, are described, for example, in the patent applications WO95/33197, WO 95/33198, WO 96/35940, WO 98/09156, WO 99/40415, PCT/EP00/04869, and PCT/EP 01/00605.

In U.S. Pat. Nos. 5,820,822, 5,232,984, 5,380,556, 6,231,892, 5,462,990,5,627,223, and 5,849,839 graft copolymers are described which comprise acharged, poly-ionic main chain and bound thereto (“grafted,”)“non-interactive” (adsorption-resistant, uncharged) side chains. Forexample, the production of so-called “bio-compatible” surfaces ofso-called “micro-capsules” to be applied in vivo or of implants isdescribed as application of such polymers. Thereby, the term“bio-compatibility” is applied in the meaning of the ability ofpreventing or, at least, minimizing the adhesion of cells or proteins tosuch coated surfaces, which could, e.g., lead to an immune defense or toa final rejection of an implant in a living organism. This property isachieved upon promoting by electrostatic interaction the adhesion of thecharged polymer main chain to an oppositely charged surface of thecarrier to be coated, and enabling the adhesion of biomolecules by meansof the “non-interactive” (uncharged) side chains.

Applications of such polymer coatings in bio-analytics, e.g. for theproduction of an adhesion-promoting layer for the immobilization ofbiological recognition elements on a sensor platform, are described inWO 00/65352. Here poly(L-lysine)-g-poly(ethyleneglycol) (PLL-g-PEG) ispreferred as a graft co-polymer. In this context, g annotates thegrafting ratio, i.e. the ratio between the number of lysine units andthe number of polyethylene glycol side chains (“PEG” side chains).

As mentioned in WO 00/65352, the optimum value of g is always dependenton the size of the PEG side chains and the application underconsideration. Optimum values of 3<g<10, preferably of 4<g<7, for PEGchains with a molecular weight of 5000 Da, and of 2<g<8, preferably of3<g<5, for a PEG molecular weight of 2000 Da, are specified in WO00/65352. These values in WO 00/65352 are related to the minimization ofnonspecific binding to a surface coated with PLL-g-PEG, the surfacebeing dedicated for the detection of proteins by means of sensorswhereon the analyte-specific recognition elements had been immobilizedon a PLL-g-PEG coated surface.

Surprisingly, it has now been found that optimum ratios between specificand non-specific binding (or specific and non-specific hybridization,respectively), for the detection of nucleic acids in nucleic acidhybridization assays using nucleic acids, immobilized as recognitionelements (here also called “capture probes”) on a surface coated withPLL-g-PEG, are achieved at average values of g between 7 and 13.

Therefore, a first subject of the invention is a surface for theimmobilization of one or several first nucleic acids as recognitionelements for the production of a recognition surface for the detectionof one or several second nucleic acids in one or more samples which arebrought into contact with the recognition surface, the first nucleicacids being applied to a layer of PLL-g-PEG as a surface forimmobilization, characterized in that the grafting ratio g has anaverage value between 7 and 13.

Thereby it is preferred that the grafting ratio g has a medium valuebetween 8 and 12.

It is preferred simultaneously that the molecular weight of thepolyetheyleneglycol side chains (“PEG” side chains) is between 500 Daand 7000 Da. Especially preferred is if the molecular weight of the PEGside chains is between 1500 Da and 5000 Da.

Preferably, the surface for the immobilization of one or several firstnucleic acids, according to the invention, is deposited on a solidcarrier. This carrier is preferably essentially optically transparent.

The term “essentially optically transparent” is understood to mean thatcarriers or layers thus characterized are a minimum of 95% transparentat least at the wavelength of light delivered from an external lightsource for its optical path perpendicular to said carrier or layer,respectively, provided the carrier or layer is not reflecting. In thecase of partially reflecting carriers or layers, “essentially opticallytransparent” is understood to mean that the sum of transmitted andreflected light and, if applicable, light in-coupled into a carrier orlayer and guided therein amounts to a minimum of 95% of the deliveredlight at the point of incidence of the delivered light.

The essentially optically transparent carrier preferably comprises amaterial from the group comprising moldable, sprayable or millableplastics, metals, metal oxides, silicates, such as glass, quartz orceramics.

It is also preferred if the immobilization surface according to theinvention is itself essentially optically transparent.

Preferably, the immobilization surface (as a PLL-g-PEG layer) has athickness of less than 200 nm, preferably of less than 20 nm.

It is characteristic for specific embodiments that the surface forimmobilization is deposited on a solid carrier, in the surface of whichare structured recesses for generation of sample compartments. Thereby,these recesses in the surface of the carrier preferably have a depth of20 μm to 500 μm, especially preferable of 50 μm to 300 μm.

Embodiments of an immobilization surface according to the invention arepreferred, which are characterized in that the essentially opticallytransparent carrier comprises a continuous optical waveguide or anoptical waveguide divided into individual waveguiding areas. It isespecially preferred if the optical waveguide is an optical filmwaveguide with a first essentially optically transparent layer (a)facing the immobilization surface on a second essentially opticallytransparent layer (b) with a refractive index lower than that of layer(a). It is also preferred if said optical film waveguide is essentiallyplanar.

It is characteristic of such an embodiment of an immobilization surfaceon an optical film waveguide as a carrier that, for the in-coupling ofexcitation light into the optically transparent layer (a), this layer isin optical contact with one or more optical in-coupling elements fromthe group comprising prism couplers, evanescent couplers with combinedoptical waveguides with overlapping evanescent fields, butt-end couplerswith focusing lenses, preferably cylinder lenses, arranged in front ofone face of the waveguiding layer, and grating couplers.

Thereby it is preferred that the excitation light is in-coupled into theoptically transparent layer (a) using one or more grating structures (c)which are featured in the optically transparent layer (a). It is alsopreferred that out-coupling of light guided in the optically transparentlayer (a) is performed using one or more grating structures (c′) whichare featured in the optically transparent layer (a) and have the same ordifferent period and grating depth as grating structures (c).

Further planar optical film waveguides and modifications thereof whichare suitable as carriers of an immobilization surface according to theinvention are described for example in patent applications WO 95/33197,WO 95/33198, WO 96/35940, WO 98/09156, WO 99/40415, PCT/EP 00/04869 andPCT/EP 01/00605. The content of these patent applications is thereforeintroduced in its entirety as an integral part of this description.

Especially preferred are such embodiments of an immobilization surfaceaccording to the invention, wherein the nucleic acids immobilizedthereon as recognition elements are arranged in discrete (laterallyseparated) measurement areas. Up to 1,000,000 measurement areas may beprovided in a 2-dimensional arrangement, and a single measurement areamay cover an area of 10⁻⁴ mm²-10 mm². It is preferred that themeasurement areas are arranged in a density of more than 10, preferablymore than 100, especially preferably more than 1000 measurement areasper square centimeter.

The discrete (laterally separated) measurement areas may be generated onsaid immobilization surface by the laterally selective application ofnucleic acids as recognition elements, preferably using one or moremethods from the group of methods comprising ink-jet spotting,mechanical spotting by means of pin, pen or capillary, micro-contactprinting, fluidic contact of the measurement areas with the biologicalor biochemical or synthetic recognition elements through theirapplication in parallel or intersecting microchannels, upon exposure topressure differences or to electric or electromagnetic potentials, andphotochemical or photolithographic immobilization methods.

A further subject of the invention is a method for the simultaneous orsequential, qualitative and/or quantitative detection of one or moresecond nucleic acids in one or more samples, wherein said samples and ifnecessary further reagents are brought into contact with animmobilization surface according to any of the embodiments describedhereinbefore, on which surface one or several first nucleic acids areimmobilized as recognition elements for the specificbinding/hybridization with said second nucleic acids, and changes inoptical or electronic signals resulting from the binding/hybridizationwith these second nucleic acids or of further tracer substances used foranalyte detection are measured.

It is preferred that the one or more samples are pre-incubated with amixture of the various tracer reagents for determining the secondnucleic acids to be detected in said samples, and these mixtures arethen brought into contact with the first nucleic acids immobilized on animmobilization surface according to the invention in a single additionstep. Thereby it is preferred that the detection of the one or moresecond nucleic acids is based on the determination of the change in oneor more luminescences.

There are different optical excitation configurations which can beapplied for luminescence excitation. One possibility consists indelivering the excitation light from one or more light sources, forexcitation of one or more luminescences, in an epi-illuminationconfiguration.

Characteristic for another possible configuration is that the excitationlight from one or more light sources for the excitation of one or moreluminescences is delivered in a transillumination configuration.

Such an embodiment of the method according to the invention is preferredwherein the immobilization surface is arranged on an optical waveguidewhich is preferably essentially planar, wherein one or more samples withsecond nucleic acids to be detected therein and, if necessary furthertracer reagents, are brought sequentially or in a single addition stepafter mixture with said tracer reagents, into contact with said firstnucleic acids immobilized as recognition elements on an immobilizationsurface according to the invention, and wherein the excitation lightfrom one or more light sources is in-coupled into the optical waveguideusing one or more optical coupling elements from the group comprisingprism couplers, evanescent couplers with combined optical waveguideswith overlapping evanescent fields, butt-end couplers with focusinglenses, preferably cylinder lenses, arranged in front of one face of thewaveguiding layer, and grating couplers.

Characteristic for another preferred embodiment of the method accordingto the invention is that the detection of one or more second nucleicacids is performed on a grating structure (c) or (c′) formed in thelayer (a) of an optical film waveguide, based on changes in theresonance conditions for the in-coupling of excitation light into layer(a) of a carrier formed as film waveguide or for out-coupling of lightguided in layer (a), these changes resulting from binding/hybridizationof said second nucleic acids or further tracer reagents to the firstnucleic acids immobilized as recognition elements in the region of saidgrating structure on an immobilization surface according to theinvention.

It is especially preferred if said optical waveguide is provided as anoptical film waveguide with a first optically transparent layer (a) on asecond optically transparent layer (b) with lower refractive index thanlayer (a), wherein excitation light is further in-coupled into theoptically transparent layer (a) with the aid of one or more gratingstructures, which are featured in the optically transparent layer (a),and delivered as a guided wave to measurement areas (d) located thereon,and wherein the luminescence of molecules capable of luminescence,generated in the evanescent field of said guided wave, is furtherdetermined using one or more detectors, and the concentration of one ormore nucleic acids to be detected is determined from the intensity ofthese luminescence signals.

Thereby, (1) the isotropically emitted luminescence or (2) luminescencein-coupled into the optically transparent layer (a) and out-coupled viagrating structure (c) or (c′) or, simultaneously, luminescences of both(1) and (2) may be measured.

It is preferred that a luminescence dye or luminescent nanoparticle isused as luminescence label for luminescence generation, which label canbe excited and emits at a wavelength between 300 nm and 1100 nm.

The luminescence label may be bound to the second nucleic acidsthemselves to be detected as analytes or, in a competitive assay, tonucleic acids with the same sequence as said second nucleic acids to bedetected and added to the sample as competitors at a knownconcentration, or, in a multistep assay, to one of the binding partnersof the first nucleic acids immobilized as recognition elements, or tosaid immobilized first nucleic acids themselves. As a multi-step assayis is here understood that not only a single second nucleic acid (as theanalyte) with a sequence at least partially complementary to thesequence of the corresponding first nucleic acid is bound or hybridized,respectively, to the immobilized first nucleic acids, but that, forexample, further nucleic acids are bound to these second nucleic acids.

It is characteristic for special embodiments of the method according tothe invention that a second luminescence label or further luminescencelabels are used with excitation wavelengths either the same as ordifferent from that of the first luminescence label and the same ordifferent emission wavelength. Such embodiments may be designed in sucha way, upon the corresponding selection of the spectral properties ofthe applied luminescence labels, that the second or further luminescencelabels can be excited at the same wavelength as the first luminescencelabel, but emit at different wavelengths.

For certain applications, for example for measurements independent ofeach other, applying different excitation and detection labels, it isadvantageous if the excitation spectra and emission spectra of theluminescence dyes used overlap only little or not at all.

For another special embodiment of the method it is characteristic thatcharge or optical energy transfer from a first luminescence labelserving as donor to a second luminescence label serving as acceptor isused for the purpose of detecting the second nucleic acids as analytes.

Characteristic for another special embodiment of the method according tothe invention is that changes in the effective refractive index on themeasurement areas are determined in addition to the determination of oneor more luminescences.

It is advantageous if the one or more luminescences and/ordeterminations of light signals at the excitation wavelength are carriedout using a polarization-selective procedure. It is especially preferredif the one or more luminescences are measured at a polarizationdifferent from that of the excitation light.

The method according to the invention is characterized in that thesamples to be analyzed may be aqueous solutions, especially buffersolutions, or naturally occurring body fluids such as blood, serum,plasma, urine or tissue fluids. A sample to be analyzed may also be anoptically turbid fluid, surface water, a soil or plant extract, abiological or synthetic process broth. The samples to be analyzed mayalso be prepared from biological tissue parts or cells.

A further subject of the invention is the use of an immobilizationsurface according to the invention and/or a method according to theinvention for quantitative or qualitative analyses in screening methodsin pharmaceutical research, clinical and pre-clinical development, forreal-time binding studies and the determination of kinetic parameters inaffinity screening and in research, for qualitative and quantitativeanalyte determinations, especially for DNA and RNA analytics and for thedetermination of genomic or proteomic differences in the genome, such assingle nucleotide polymorphisms, for the measurement of protein-DNAinteractions, for the determination of control mechanisms for mRNAexpression and for protein (bio)synthesis, for the generation oftoxicity studies and the determination of expression profiles,especially for the determination of biological and chemical markercompounds, such as mRNA, pathogens or bacteria in pharmaceutical productresearch and development, human and veterinary diagnostics, agrochemicalproduct research and development, for symptomatic and pre-symptomaticplant diagnostics, for patient stratification in pharmaceutical productdevelopment and for therapeutic drug selection, for the determination ofpathogens, nocuous agents and germs, especially of salmonella, prions,viruses and bacteria, especially in food and environmental analytics.

The invention will be further explained by the following example.

EXAMPLE

1. Chemicals

1.1. Buffer Solutions

The following buffer solutions were used:

Buffer 1:

-   4×SSC (600 mM NaCl 160 mM sodium citrate, pH 7.5)    Buffer 2:-   4×SSC (600 mM NaCl/60 mM sodium citrate, pH 7.5) comprising 50%    formamide    Washing Buffer 1:-   1×SSC (150 mM NaCl/15 mM sodium citrate, pH 7.5) comprising 0.1% SDS    Washing Buffer 2:-   0.1×SSC (15 mM NaCl/1.5 mM sodium citrate, pH 7.5) comprising 0.1%    SDS    Washing Buffer 3:-   0.1×SSC (15 mM NaCl/1.5 mM sodium citrate, pH 7.5)    1.2. First Nucleic Acids to be Immobilized

A mouse brain “longmer set”, derived from 96 genes (Lion Bioscience,Heidelberg, Germany), representing low to medium expressed genes frombrain tissue of a mouse, were used, being provided as oligonucleotidesof a length of 70 nucleotides each (“longmers”), the sequence of whichwas selected by Operon (Alamada, Calif., USA) from the sequences of saidgenes and was also produced by Operon.

1.3. Second Nucleic Acids to be Detected as Analyte

Starting from mouse brain, the total RNA was isolated using the kitRNeasy (QIAGEN, Hilden, Germany). In a further step, mRNA was isolatedfrom this isolate of total RNA using the kit Oligotex (QIAGEN, Hilden,Germany). Then the mRNA isolate was utilized as a template for reversetranscription (by means of Reverse Transcriptase Omniscript, QIAGEN,Hilden, Germany). Using a poly (dT) primer, all mRNA molecules with apoly (dA) tail were transcribed to cDNA. Nucleotides fluorescentlylabeled with Cy5 (Amersham, Arlington Heights, USA) were applied forthis transcription step, resulting in fluorescently labeled cDNA.

Dependent on the yield of mRNA isolation and the efficiency of reversetranscription, the labeled cDNA does represent the whole variety of mRNAexpressed in the mouse brain used.

1.4. Production of poly(L-lysine)-g-poly(ethyleneglycol) (PLL-g-PEG)

Materials

Poly(L-lysine) hydrobromide (molecular weight about 20 kDa) was obtainedfrom Sigma-Aldrich (Buchs, switzerland). The N-hydroxy succinimidylester of methoxy poly(ethyleneglycol) propionic acid (MeO-PEG-SPA,molecular weight 2 kDA) was obtained from Shearwater Polymers Inc.(Huntsville, USA). 4-(2-hydroxyethyl) piperazine-1-ethane sulfonic acid(HEPES) and further chemicals for the preparation of buffers werepurchased from Fluka (Buchs, Switzerland).

All aqeuous solutions were produced using ultra-pure water (18 MOhm cm)from an “Easy Pure reverse Osmosis System” (Barnstead Thermolyne,Dubuque, USA).

Synthesis of PLL-G-PEG

The synthesis of PLL-g-PEG has been described by Sawhney and Hubbell (A.S. Sawhney, J. A. Hubbell, Biomaterials 13 (1992) 863-870). The studieswhich serve as a basis for the present application used procedures basedon a method developed by Elbert and Hubbell (D. L. Elbert, J. A.Hubbell, J. Biomed. Mater. Res. 42 (1998) 55-65).

N-Hydroxysuccinimidyl ester of poly(ethyleneglycol) (“PEG”) is reactedwith poly(L-lysine) (“PLL”) under stoichiometric conditions tomanufacture the desired product. The details on this synthesis aredescribed hereinafter.

The nomenclature used hereinafter to describe the various PLL-g-PEGderivatives includes the molecular weights of the polymer sub-chains ofthe copolymers and the grafting ratio. Accordingly,“PLL(20)-g[3.5]-PEG(2)” describes a polymer composed of a main chain ofpoly(L-lysine) with a molecular weight of 20 kDa and side chainscomprising poly(ethyleneglycol) with a molecular weight of 2 kDa. Thegrafting ratio of 3.5 means that, on average, PEG chains in each caseare bound to two of seven lysine groups (lysine units). Since all thepolymers mentioned in this example were manufactured from identicalprecursor products, the abbreviation “PLL-g[3,7]-PEG” is also to be usedas an alternative to “PLL(20)-g[3,7]-PEG(2)”.

Poly(L-lysine)hydrobromide (“PLL-HBr”) is dissolved in 25 ml sodiumtetraborate buffer (“STBB”, 50 mM, pH 8.5) per gram PLL-HBr. Thesolution is stirred, then filtered (0.22 μm Durapore membrane, sterileMillex GV, Sigma-Aldrich, Buchs, Switzerland) and filled into a sterileculture tube. While the solution is constantly stirred, a suitablequantity of MeO-PEG-SPA powder is then added according to stoichiometricconditions. After a further six hours of stirring, the solution istransferred at room temperature to a dialysis tube (Spectr/Por dialysistubes, molecular weight cut-off 6-8 kDa, Sochochim, Lausanne,Switzerland). The dialysis is carried out for 24 hours in a liter ofphosphate-buffered saline (“PBS”, 10 mM, pH 7.0), followed by a another24 hours of further dialysis in a liter of deionized water. The productis then lyophilized for 48 hours.

The control of the grafting ratio is performed using 1H-NMR. 6 differentpolymers with grafting ratios of 3.7, 7.4, 8.4, 9.0, 11.8, and 13.0 areproduced as described hereinbefore.

2. Carrier

As a carrier of an immobilization surface according to the invention aplanar optical film waveguide is used with the external dimensions of 57mm in width (parallel to the grating lines of a grating structure (c)modulated in layer (a) of the film waveguide)×14 mm in length(perpendicular to the grating structures)×0.7 mm in height. 6 microflowcells can be created in the pattern of part of a column of a standardmicrotiter plate (9 mm spacing) by combination with a polycarbonateplate featuring open cavities in the direction of the sensor platformwith the internal dimensions of 5 mm wide×7 mm long×0.15 mm high, eitherdirectly on the surface of layer (a) or after deposition of furtherlayers, especially of an immobilizattion surface according to theinvention, on layer (a). The polycarbonate plate may be adhered to thecarrier in such a way that the cavities are then tightly sealed againsteach other. This polycarbonate plate is constructed such that it can bejoined together with a substrate (“meta-carrier”) with the basicdimensions of standard microtiter plates in such a way that the pitch(arrangement of rows or columns) of the inlets of the flow cells matchesthe pitch of the wells of a standard microtiter plate.

The substrate material (optically transparent layer (b) of the planaroptical film waveguide as a carrier) comprises AF 45 glass (refractiveindex n=1.52 at 633 nm). The substrate features a pair of in-couplingand out-coupling gratings with grating lines (318 nm period) runningparallel with the width of the sensor platform at a grating depth of12±3 nm, wherein the grating lines are drawn over the whole width of thefilm waveguide. The distance between the two consecutive gratings is 9mm, and the length of the individual grating structures (parallel withthe length of the sensor platform) is 0.5 mm. The distance between thein-coupling and out-coupling grating of a grating pair is selected suchthat the excitation light in each case can be in-coupled within theregion of the sample compartments, after combination of the sensorplatform with the aforementioned polycarbonate plate, whereas theout-coupling takes place outside the region of the sample compartment.The wave-guiding, optically transparent layer (a) comprising Ta₂O₅ onthe optically transparent layer (b) has a refractive index of 2.15 at633 nm (layer thickness 150 nm).

To prepare for immobilization of the biochemical or biological orsynthetic recognition elements, the optical film waveguide as a carrieris cleaned using organic and inorganic reagents (e.g. propanol andsulfuric acid, with intermediate washing steps with water) in anultrasonication device.

3. Generation of the Immobilization Surface

A solution of PLL-g-PEG in PBS buffer (1 mg/ml) is produced and filteredthrough 0.22 μm Durapore menbranes. Instead of PBS buffer, for example,also HEPES buffer can be used. 570 μl of the PLL-g-PEG solution arepipetted into a special incubation chamber for the coating of thecarrier as described in section 2. of this example. Then the carriersare inserted into the incubation chamber in such a way that the surfaceto be coated, i.e. the surface of the layer (a) on the example of aplanar optical film waveguide as a carrier to be coated, gets intocontact with the polymer solution. After a two-hours incubation at roomtemperature, the coated carriers are rinsed with ultra-pure water andblown dry with nitrogen.

4. Immobilization of the First Nucleic Acid/Generation of DiscreteMeasurement Areas

The 96 oligonucleotides with a length of 70 nucleotides each, at aconcentration of 40 μM in 10 mM carbonate buffer (pH 9.2, with anaddition of 5% DMSO), as described in section 1.2, are deposited asbiological recognition elements on the immobilization surface generatedas described above using a commercial spotter (GMS 417 Arrayer,Affymetrix, Santa Clara. CA, USA) and incubated over night. The distancebetween the measurement areas (spots) thus generated is 340 μm. In onearray always two spots with identical base sequence are generated, asingle array thus comprising 192 spots. Up to 6 similar arrays aregenerated on a film waveguide as a carrier, according to section 2.

Arrays of immobilized first nucleic acids are generated in a similarmanner on the six carriers with immobilization surfaces of differentgrafting ratio.

The polycarbonate plate described above is joined with the carriercoated with the immobilization surface, comprising the first nucleicacids deposited on the immobilization surface, in such a way that theindividual sample compartments are fluidically sealed against oneanother and the generated “longmer” arrays, together with thecorresponding in-coupling grating (c), are arranged each within one ofthe 6 sample compartments.

5. Hybridization Assay as an Integral Part of the Method According tothe Invention for the Determination of One or More Second Nucleic Acids

The carrier provided with discrete measurement areas on a depositedimmobilization surface according to the invention, and provided as aplanar optical film waveguide, joined with a polycarbonate plate forgeneration of 6 sample compartments (“chambers”) according to section 2,of this example, is inserted into a “meta-carrier”. For purposes ofmoistening/equilibration the two-dimensional arrangements of measurementarrays (“microarrays”) are filled with 90 μl buffer 1.

A sample of the second nucleic acids (“target probe”) for hybridizationto be detected as analyte is prepared from labeled cDNA (according tosection 1.3) at an amount corresponding to 25 ng mRNA. An amount of cDNAin 50 μl hybridization buffer (buffer 2), corresponding to an amount of25 ng mRNA, is added by pipetting. For purposes of denaturation, thetarget probe is heated to 95° C. for 5 min and then stored on ice for 5min. Buffer 1 is evacuated from the chambers, and the target probe ispipetted upon avoiding air bubbles.

For hybridization, the “meta carrier” is inserted into a thermocycler(MJ Research PCT-200 with an adapter plate) for 35 min at 75° C. (stepof denaturation) and incubated then for 18 hours at 42° C.(hybridization step).

After termination of the hybridization, the following washing steps areperformed: The chambers are evacuated by application of vacuum, thenfilled with 90 μl buffer 1 and then temperature-equilibrated at roomtemperature in the “meta carrier”.

Then the chambers are evacuated again, filled with 90 μl washing buffer1 and incubated for 7 min at room temperature. In a similar way,evacuation and filling is repeated using once washing buffer 2 and twicewashing buffer 3, Finally, the chambers are evacuated and filled withbuffer 1.

The hybridization assay as described above is performed in a similar waywith all 6 carriers comprising immobilization surfaces of differentgrafting ratios.

6. Analytical System and Measurement Method for the Detection of One orMore Analytes

The excitation light from a laser diode with emission at 635 nm isexpanded to a ray bundle of slit-type cross section (perpendicular tothe optical axis) using a lens system comprising a cylindrical lens anda diaphragm, the size of the ray bundle in the cross-section of lightirradiated onto the planar optical film waveguide, in parallel to thegrating lines, corresponding almost exactly to the section of thein-coupling grating located within a sample compartment.

The angle between the incoming, parallel excitation light bundle and theplane of the planar optical film waveguide is adjusted to the resonanceangle for maximum in-coupling into the waveguiding layer (a) (−110), aswell as the corresponding optimum position of the excitation light to bein-coupled on the in-coupling grating (first grating). This optimizationis performed in an automated manner, wherein the light out-coupled bythe second grating located outside of the sample compartment is directedto a photodiode, the signal of which is amplified in an adequate way andwherein the photodiode signal is optimized to a maximum value, based onthe principle of a “feedback loop”, upon further adjustments of thecarrier with respect to the coupling angle and the lateral position.

Light emanating from the microarray, from the region of the measurementsurface within a sample compartment on the carrier provided as a planaroptical film waveguide (image area about 6 mm×8 m), is collected by atandem objective and focussed onto a CCD camera comprising a CCD chip(active area about 5 mm×7 mm with 766 pixels, pixel size: 9 μm).Dependent on the imaging system, this configuration enables a lateralresolution of about 10 μm to 20 μm.

An interference filter (670 DF 40, Omega Optical, Brattleborough, Vt.,USA) is positioned between the two halves of the tandem objective, in anessentially parallel (i.e. less than 10° divergent or convergent) partof the emission ray path, for collection of the light emanating from thearray at the fluorescence wavelength of the applied fluorescence label(Cy 5).

After accomplished hybridization of the immobilized first nucleic acidswith the second, fluorescently labeled nucleic acids supplied as thesample, in each case the emission light from all measurement areaslocated within a sample compartment is collected as one image by acooled CCD camera.

7. Analysis of the Measurement Data

The medium signal intensity from the measurement areas, for the bindingand detection of analyte molecules due to a potentially generatedfluorescence of fluorescence labels (Cy5 according to the example inhand) is determined using image analysis software.

The raw data obtained from the individual pixels of the camera form atwo-dimensional matrix of the digitized measurement data, with themeasured intensity as the measurement value of a pixel corresponding tothe surface section of the sensor platform imaged onto said pixel. Fordata analysis, at the beginning a two-dimensional (coordinate) net issuperimposed over the image points (pixel values) in such a way thateach spot is contained in an individual, two-dimensional net element.Within this net element, an “analysis element” (area of interest, “AOI”)is assigned to each spot, with a geometry optimized for matching thespot geometry. These AOIs can have any geometric form, for examplecircular form. The location of the AOIs in the two-dimensional net isindividually optimized as a function of the signal intensity recorded bythe corresponding pixels. Dependent on the definitions set by the user,the initially defined radius of an AOI can be preserved or can bere-adjusted according to the geometry and size of a given spot. Forexample, the arithmetic average of the pixel values (signal intensities)can be determined as the mean gross signal intensity of every spot.

The background signals are determined from the signal intensitiesmeasured between the spots. For this purpose, for example, furthercircles can be defined, which are concentric with a given circular spot(and the assigned “spot AOI”), but have a larger radius. Of course, theradii of these concentric circles have to be smaller than the distancebetween adjacent spots. Then, for example, the region between the “spotAOI” and the first larger concentric circle can be disregarded, and theregion between said first larger and a second still larger concentriccircle can be defined as the AOI for the background determination(“background AOI”). It is also possible to define regions betweenadjacent spots, preferably located in the middle between adjacent spots,as AOIs for the determination of the background signal intensities. Fromthese signal values the average background signal can then be determinedin analogous way as described above, for example as the arithmeticaverage of the pixel values (signal intensities) of the chosen“background AOI”. The average net signal intensity can then bedetermined as the difference between the local average gross and thelocal average background signal intensity.

8. Results

For all 6 carriers with immobilization surfaces of different graftingratio g, the fluorescence signals from the measurement areas (“spots”)of the arrays were measured in the analytical system after terminationof the hybridization assays (according to section 6. of this example).Images of the fluorescence signals determined for 4 g values, namely3.7, 7.4, 9.0, and 11.8, are shown in FIG. 1 a-1 d. For thedetermination of the net fluorescence signals, as the difference betweenthe gross fluorescence signals (arithmetic mean of the pixel values inthe AOIs) and the background signals, according to section 7 of thisexample, the signals from two spot pairs (duplicates) each, as anexample for the fluorescence signals after hybridization with cDNA fromhighly expressed genes (spot group I in FIG. 1 a-d), weaker expressedgenes (spot group II in FIG. 1 a-d), were analyzed (marked in FIG. 1a-d). The strong effect of the grafting ratio g on the signalintensities is already evident from the comparison of images 1 a-1 d,which are all displayed in the same dynamic range: Many spots areclearly visible in FIG. 1 d (g=1.8), visible in FIG. 1 c (g=9.0), hardlyvisible in FIG. 1 b (g=7.4), and not visible in FIG. 1 a (g=3.7). Thisconcerns, for example, the complete left row of spots.

The calculated net fluorescence intensities, as average values of thesignals from always two spots of similar type, are displayed in FIG. 2as a function of the grafting ratio. For both selected spot pairs, thefluorescence signals are relatively low in the region of g=3.7 to g=7.4.Starting from g=8.4, a strong increase of the fluorescence signals isobserved. With further increase of g, am extended flat region of highsignal intensities (“plateau”) is reached, before the signal intensitiesdecrease at values of g>11.8.

Based on these results it is concluded that for immobilization surfacesof the kind at hand, for achieving net signals as high as possible inhybridization assays as described herein, the grafting ratio g shouldhave a value between 7 and 13, preferably between 8 and 12.

1. A surface for the immobilization of one or several first nucleicacids as recognition elements (“immobilization surface”), for theproduction of a recognition surface for the detection of one or severalsecond nucleic acids in one or more samples which are brought intocontact with the recognition surface, the first nucleic acids beingapplied to a layer of PLL-g-PEG (graft copolymerpoly(L-lysine)-g-poly(ethyleneglycol)) as a surface for immobilization,characterized in that the grafting ratio g, in other words the ratiobetween the number of lysine units and the number of polyethylene glycolside chains (“PEG” side chains) has an average value between 7 and 13.2. A surface for the immobilization of one or several first nucleicacids according to claim 1, wherein the grafting ratio g has a mediumvalue between 8 and
 12. 3. A surface for the immobilization of one orseveral first nucleic acids according to claim 1, wherein the molecularweight of the polyetheyleneglycol side chains (“PEG” side chains) isbetween 500 Da and 7000 Da.
 4. A surface for the immobilization of oneor several first nucleic acids according to claim 1, wherein themolecular weight of the polyetheyleneglycol side chains (“PEG” sidechains) is between 1500 Da and 5000 Da.
 5. A surface for theimmobilization of one or several first nucleic acids according to claim1, wherein said surface is deposited on a solid carrier.
 6. A surfacefor the immobilization of one or several first nucleic acids accordingto claim 5, wherein said solid carrier is an essentially opticallytransparent carrier.
 7. A surface for the immobilization of one orseveral first nucleic acids according to claim 6, wherein theessentially optically transparent carrier comprises a material from thegroup comprising moldable, sprayable or millable plastics, metals, metaloxides, silicates, such as glass, quartz or ceramics.
 8. A surface forthe immobilization of one or several first nucleic acids according toclaim 1, wherein said surface is essentially optically transparent.
 9. Asurface for the immobilization of one or several first nucleic acidsaccording to claim 1, wherein said surface (as a PLL-g-PEG layer) has athickness of less than 200 nm, preferably of less than 20 nm.
 10. Animmobilization surface according to claim 6, wherein said surface forimmobilization is deposited on a solid carrier, in the surface of whichare structured recesses for generation of sample compartments.
 11. Animmobilization surface according to claim 10, wherein said recesses inthe surface of the carrier have a depth of 20 μm to 500 μm, especiallypreferably 50 μm to 300 μm.
 12. An immobilization surface according toclaim 6, wherein the essentially optically transparent carrier comprisesa continuous optical waveguide or an optical waveguide divided intoindividual waveguiding areas.
 13. An immobilization surface according toclaim 12, wherein the optical waveguide is an optical film waveguidewith a first essentially optically transparent layer (a) facing theimmobilization surface on a second essentially optically transparentlayer (b) with a refractive index lower than that of layer (a).
 14. Animmobilization surface according to claim 13, wherein said optical filmwaveguide is essentially planar.
 15. An immobilization surface accordingto claim 13, wherein, for the in-coupling of excitation light into theoptically transparent layer (a), this layer is in optical contact withone or more optical in-coupling elements from the group comprising prismcouplers, evanescent couplers with combined optical waveguides withoverlapping evanescent fields, butt-end couplers with focusing lenses,preferably cylinder lenses, arranged in front of one face of thewaveguiding layer, and grating couplers.
 16. An immobilization surfaceaccording to claim 15, wherein the excitation light is in-coupled intothe optically transparent layer (a) using one or more grating structures(c) which are featured in the optically transparent layer (a).
 17. Animmobilization surface according to claim 15, wherein light guided inthe optically transparent layer (a) is out-coupled using one or moregrating structures (c′) which are featured in the optically transparentlayer (a) and have the same or different period and grating depth asgrating structures (c).
 18. An immobilization surface according to claim1, wherein the nucleic acids immobilized thereon as recognition elementsare arranged in discrete (laterally separated) measurement areas.
 19. Animmobilization surface according to claim 18, wherein up to 1,000,000measurement areas are provided in a 2-dimensional arrangement and asingle measurement area covers an area of 10⁻⁴ mm²-10 mm².
 20. Animmobilization surface according to claim 18, wherein the measurementareas are arranged in a density of more than 10, preferably more than100, especially preferably more than 1000 measurement areas per squarecentimeter.
 21. An immobilization surface according to claim 18, whereindiscrete (laterally separated) measurement areas are generated on saidimmobilization surface by the laterally selective application of nucleicacids as recognition elements, preferably using one or more methods fromthe group of methods comprising ink-jet spotting, mechanical spotting bymeans of pin, pen or capillary, micro-contact printing, fluidic contactof the measurement areas with the biological or biochemical or syntheticrecognition elements through their application in parallel orintersecting microchannels, upon exposure to pressure differences or toelectric or electromagnetic potentials, and photochemical orphotolithographic immobilization methods.
 22. A method for thesimultaneous or sequential, qualitative and/or quantitative detection ofone or more second nucleic acids in one or more samples, wherein saidsamples and if necessary further reagents are brought into contact withan immobilization surface according to claim 1, on which surface one orseveral first nucleic acids are immobilized as recognition elements forthe specific binding/hybridization with said second nucleic acids, andchanges in optical or electronic signals resulting from thebinding/hybridization with these second nucleic acids or of furthertracer substances used for analyte detection are measured.
 23. A methodaccording to claim 22, wherein the one or more samples are pre-incubatedwith a mixture of the various tracer reagents for determining the secondnucleic acids to be detected in said samples, and these mixtures arethen brought into contact with the first nucleic acids immobilized onsaid immobilization surface in a single addition step.
 24. A methodaccording to claim 22, wherein the detection of the one or more secondnucleic acids is based on the determination of the change in one or moreluminescences.
 25. A method according to claim 22, wherein theexcitation light from one or more light sources for the excitation ofone or more luminescences is delivered in an epi-illuminationconfiguration.
 26. A method according to claim 22, wherein theexcitation light from one or more light sources for the excitation ofone or more luminescences is delivered in a transilluminationconfiguration.
 27. A method according to one of claims 22-24 claim 22,wherein the immobilization surface is arranged on an optical waveguidewhich is preferably essentially planar, wherein one or more samples withsecond nucleic acids to be detected therein and, if necessary furthertracer reagents, are brought sequentially or in a single addition stepafter mixture with said tracer reagents, into contact with said firstnucleic acids immobilized as recognition elements on said immobilizationsurface, and wherein the excitation light from one or more light sourcesis in-coupled into the optical waveguide using one or more opticalcoupling elements from the group comprising prism couplers, evanescentcouplers with combined optical waveguides with overlapping evanescentfields, butt-end couplers with focusing lenses, preferably cylinderlenses, arranged in front of one face of the waveguiding layer, andgrating couplers.
 28. A method according to claim 27, wherein thedetection of one or more second nucleic acids is performed on a gratingstructure (c) or (c′) formed in the layer (a) of an optical filmwaveguide, based on changes in the resonance conditions for thein-coupling of excitation light into layer (a) of a carrier formed asfilm waveguide or for out-coupling of light guided in layer (a), thesechanges resulting from binding/hybridization of said second nucleicacids or further tracer reagents to the first nucleic acids immobilizedas recognition elements in the region of said grating structure on saidimmobilization surface.
 29. A method according to claim 27, wherein saidoptical waveguide is designed as an optical film waveguide with a firstoptically transparent layer (a) on a second optically transparent layer(b) with lower refractive index than layer (a), wherein excitation lightis further in-coupled into the optically transparent layer (a) with theaid of one or more grating structures, which are featured in theoptically transparent layer (a), and delivered as a guided wave tomeasurement areas (d) located, and wherein the luminescence of moleculescapable of luminescence, generated in the evanescent field of saidguided wave, is further determined using one or more detectors, and theconcentration of one or more nucleic acids to be detected is determinedfrom the intensity of these luminescence signals.
 30. A method accordingto claim 29, wherein (1) the isotropically emitted luminescence or (2)luminescence in-coupled into the optically transparent layer (a) andout-coupled via grating structure (c) or (c′) or luminescences of both(1) and (2) are measured simultaneously.
 31. A method according to claim29, wherein, for the generation of luminescence, a luminescence dye orluminescent nanoparticle is used as a luminescence label, which can beexcited and emits at a wavelength between 300 nm and 1100 nm.
 32. Amethod according to claim 31, wherein the luminescence label is bound tothe second nucleic acids themselves to be detected as analytes or, in acompetitive assay, to nucleic acids with the same sequence as saidsecond nucleic acids to be detected and added to the sample ascompetitors at a known concentration, or, in a multistep assay, to oneof the binding partners of the first nucleic acids immobilized asrecognition elements, or to said immobilized first nucleic acids.
 33. Amethod according to claim 31, wherein a second luminescence label orfurther luminescence labels are used with excitation wavelengths eitherthe same as or different from that of the first luminescence label andthe same or different emission wavelength.
 34. A method according toclaim 33, wherein the second or further luminescence labels can beexcited at the same wavelength as the first luminescence label, but emitat different wavelengths.
 35. A method according to claim 33, whereinthe excitation spectra and emission spectra of the luminescence dyesused overlap only little or not at all.
 36. A method according to claim33, wherein charge or optical energy transfer from a first luminescencelabel serving as donor to a second luminescence label serving asacceptor is used for the purpose of detecting the second nucleic acidsas analytes.
 37. A method according to claim 29, wherein changes in theeffective refractive index on the measurement areas are determined inaddition to the determination of one or more luminescences.
 38. A methodaccording to claim 29, wherein the one or more luminescences and/ordeterminations of light signals at the excitation wavelength are carriedout using a polarization-selective procedure.
 39. A method according toclaim 29, wherein the one or more luminescences are measured at apolarization different from that of the excitation light.
 40. A methodaccording to claim 22, wherein the samples to be analyzed are aqueoussolutions, especially buffer solutions, or naturally occurring bodyfluids such as blood, serum, plasma, urine or tissue fluids.
 41. Amethod according to claim 22, wherein the sample to be analyzed is anoptically turbid fluid, surface water, a soil or plant extract, abiological or synthetic process broth.
 42. A method according to claim22, wherein the samples to be analyzed are prepared from biologicaltissue parts or cells.
 43. Use of an immobilization surface according toclaim 1 for quantitative or qualitative analyses in screening methods inpharmaceutical research, clinical and pre-clinical development, forreal-time binding studies and the determination of kinetic parameters inaffinity screening and in research, for qualitative and quantitativeanalyte determinations, especially for DNA and RNA analytics and for thedetermination of genomic or proteomic differences in the genome, such assingle nucleotide polymorphisms, for the measurement of protein-DNAinteractions, for the determination of control mechanisms for mRNAexpression and for protein (bio)synthesis, for the generation oftoxicity studies and the determination of expression profiles,especially for the determination of biological and chemical markercompounds, such as mRNA, pathogens or bacteria in pharmaceutical productresearch and development, human and veterinary diagnostics, agrochemicalproduct research and development, for symptomatic and pre-symptomaticplant diagnostics, for patient stratification in pharmaceutical productdevelopment and for therapeutic drug selection, for the determination ofpathogens, nocuous agents and germs, especially of salmonella, prions,viruses and bacteria, especially in food and environmental analytics.44. An immobilization surface according to claim 8, wherein said surfacefor immobilization is deposited on a solid carrier, in the surface ofwhich are structured recesses for generation of sample compartments. 45.An immobilization surface according to claim 9, wherein said surface forimmobilization is deposited on a solid carrier, in the surface of whichare structured recesses for generation of sample compartments.
 46. Animmobilization surface according to claim 8, wherein the essentiallyoptically transparent carrier comprises a continuous optical waveguideor an optical waveguide divided into individual waveguiding areas. 47.An immobilization surface according to claim 46, wherein the opticalwaveguide is an optical film waveguide with a first essentiallyoptically transparent layer (a) facing the immobilization surface on asecond essentially optically transparent layer (b) with a refractiveindex lower than that of layer (a).
 48. An immobilization surfaceaccording to claim 9, wherein the essentially optically transparentcarrier comprises a continuous optical waveguide or an optical waveguidedivided into individual waveguiding areas.
 49. An immobilization surfaceaccording to claim 48, wherein the optical waveguide is an optical filmwaveguide with a first essentially optically transparent layer (a)facing the immobilization surface on a second essentially opticallytransparent layer (b) with a refractive index lower than that of layer(a).
 50. A method for the simultaneous or sequential, qualitative and/orquantitative detection of one or more second nucleic acids in one ormore samples, wherein said samples and if necessary further reagents arebrought into contact with an immobilization surface according to claim6, on which surface one or several first nucleic acids are immobilizedas recognition elements for the specific binding/hybridization with saidsecond nucleic acids, and changes in optical or electronic signalsresulting from the binding/hybridization with these second nucleic acidsor of further tracer substances used for analyte detection are measured.51. A method according to claim 50, wherein the immobilization surfaceis arranged on an optical waveguide which is preferably essentiallyplanar, wherein one or more samples with second nucleic acids to bedetected therein and, if necessary further tracer reagents, are broughtsequentially or in a single addition step after mixture with said tracerreagents, into contact with said first nucleic acids immobilized asrecognition elements on said immobilization surface, and wherein theexcitation light from one or more light sources is in-coupled into theoptical waveguide using one or more optical coupling elements from thegroup comprising prism couplers, evanescent couplers with combinedoptical waveguides with overlapping evanescent fields, butt-end couplerswith focusing lenses, preferably cylinder lenses, arranged in front ofone face of the waveguiding layer, and grating couplers.
 52. A methodfor the simultaneous or sequential, qualitative and/or quantitativedetection of one or more second nucleic acids in one or more samples,wherein said samples and if necessary further reagents are brought intocontact with an immobilization surface according to claim 8, on whichsurface one or several first nucleic acids are immobilized asrecognition elements for the specific binding/hybridization with saidsecond nucleic acids, and changes in optical or electronic signalsresulting from the binding/hybridization with these second nucleic acidsor of further tracer substances used for analyte detection are measured.53. A method according to claim 52, wherein the immobilization surfaceis arranged on an optical waveguide which is preferably essentiallyplanar, wherein one or more samples with second nucleic acids to bedetected therein and, if necessary further tracer reagents, are broughtsequentially or in a single addition step after mixture with said tracerreagents, into contact with said first nucleic acids immobilized asrecognition elements on said immobilization surface, and wherein theexcitation light from one or more light sources is in-coupled into theoptical waveguide using one or more optical coupling elements from thegroup comprising prism couplers, evanescent couplers with combinedoptical waveguides with overlapping evanescent fields, butt-end couplerswith focusing lenses, preferably cylinder lenses, arranged in front ofone face of the waveguiding layer, and grating couplers.
 54. A methodfor the simultaneous or sequential, qualitative and/or quantitativedetection of one or more second nucleic acids in one or more samples,wherein said samples and if necessary further reagents are brought intocontact with an immobilization surface according to claim 9, on whichsurface one or several first nucleic acids are immobilized asrecognition elements for the specific binding/hybridization with saidsecond nucleic acids, and changes in optical or electronic signalsresulting from the binding/hybridization with these second nucleic acidsor of further tracer substances used for analyte detection are measured.55. A method according to claim 54, wherein the immobilization surfaceis arranged on an optical waveguide which is preferably essentiallyplanar, wherein one or more samples with second nucleic acids to bedetected therein and, if necessary further tracer reagents, are broughtsequentially or in a single addition step after mixture with said tracerreagents, into contact with said first nucleic acids immobilized asrecognition elements on said immobilization surface, and wherein theexcitation light from one or more light sources is in-coupled into theoptical waveguide using one or more optical coupling elements from thegroup comprising prism couplers, evanescent couplers with combinedoptical waveguides with overlapping evanescent fields, butt-end couplerswith focusing lenses, preferably cylinder lenses, arranged in front ofone face of the waveguiding layer, and grating couplers.
 56. Use of amethod according to claim 22 for quantitative or qualitative analyses inscreening methods in pharmaceutical research, clinical and pre-clinicaldevelopment, for real-time binding studies and the determination ofkinetic parameters in affinity screening and in research, forqualitative and quantitative analyte determinations, especially for DNAand RNA analytics and for the determination of genomic or proteomicdifferences in the genome, such as single nucleotide polymorphisms, forthe measurement of protein-DNA interactions, for the determination ofcontrol mechanisms for mRNA expression and for protein (bio)synthesis,for the generation of toxicity studies and the determination ofexpression profiles, especially for the determination of biological andchemical marker compounds, such as mRNA, pathogens or bacteria inpharmaceutical product research and development, human and veterinarydiagnostics, agrochemical product research and development, forsymptomatic and pre-symptomatic plant diagnostics, for patientstratification in pharmaceutical product development and for therapeuticdrug selection, for the determination of pathogens, nocuous agents andgerms, especially of salmonella, prions, viruses and bacteria,especially in food and environmental analytics.